Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments

• Published in: Virus research 2004-07, Vol.103 (1), p.155-161
• Main Authors: Wit, Emmie de, Spronken, Monique I.J, Bestebroer, Theo M, Rimmelzwaan, Guus F, Osterhaus, Albert D.M.E, Fouchier, Ron A.M
• Format: Article
• Language: English
• Subjects: Animals; Cell Line; Chimeric viruses; DNA, Complementary - genetics; DNA, Complementary - metabolism; Dogs; Fourth 3′ nucleotide; Humans; Influenza A virus - genetics; Influenza A virus - physiology; Influenza A/PR/8/34; Influenza virus; Plasmids; Recombination, Genetic; Reverse genetics; Transcription, Genetic; Transfection
• ISSN: 0168-1702; 1872-7492
• DOI: 10.1016/j.virusres.2004.02.028

A reverse genetics system for the generation of influenza virus A/PR/8/34 (NIBSC vaccine strain) from plasmid DNA was developed. Upon transfection of eight bidirectional transcription plasmids encoding the gene segments of A/PR/8/34 into 293T cells, virus titers in the supernatant were about 10 4 TCID 50/ml. The production of A/PR/8/34 in 293T cells was compared to that of A/WSN/33, for which virus titers in the supernatant were 10 7–10 8 TCID 50/ml. Time-course analysis of virus production indicated that the differences in virus titers were due to reinfection of 293T cells by A/WSN/33 but not A/PR/8/34. Indeed, virus titers of A/PR/8/34 comparable to those of A/WSN/33 were achieved upon addition of trypsin to the culture medium of transfected cells. The production of chimeric viruses revealed that the difference in virus titers between A/PR/8/34 and A/WSN/33 are determined primarily by differences in the surface glycoproteins hemagglutinin and neuraminidase and the polymerase protein PB1. In conclusion, high-titer virus stocks of recombinant influenza A/PR/8/34 virus can be produced as well as virus stocks with much lower titers, but without the requirement of virus amplification through replication.